There is widespread use of ionization sources (ambient and non-ambient) for a variety of applications. More recently, charged microdroplets generated by electrospray ionization and paper spray have been used to conduct chemistry at faster rates compared to bulk volumes. Uncharged droplets such as those generated by the Leidenfrost technique have also been used to explore chemistry and study the degradation of drugs in an accelerated manner. These microdroplets serve as reaction vessels in which in which some reactions are known to occur at accelerated rates. Such chemistry can be particularly useful in pharmaceutical settings to rapidly synthesize small amounts of materials in relatively short amount of time. Additionally, microdroplets may also be used to perform high throughput screening analysis. While several parameters influencing the rate of reaction in microdroplets have been explored (such as spray distance and reagent concentration), the mechanism of reaction acceleration has not been probed to a significant extent. A major portion of my dissertation describes the use of charged and uncharged microdroplets to perform quick chemistry, guide microfluidic synthesis of drugs such as diazepam, perform scale up of copper catalyzed C-O and C-N coupling reactio<a></a>ns and screen reaction conditions for pharmaceutically relevant reactions such as the Suzuki cross-coupling reaction. Additionally, work discussed here also describes development and use of existing techniques such as structured illumination microscopy to measure droplet sizes, explore the role of distances on droplet size, and study the effect of surfactants on the rate of reactions in microdroplets generated by nano-electrospray ionization. A mathematical model to understand the mechanism of increased reaction rates in microdroplets has also been presented. Additionally, this dissertation also describes ways to manipulate ions in air using various designs of 3D-printed electrodes that operate with DC potentials only and which can be easily coupled with nano-electrospray ionization sources to transmit ions over long distances

<p>The work presented here employs cryogenic ion spectroscopy for the study of protein structure, kinetics, and dynamics. The main technique used is IR-UV double resonance spectroscopy. Here peptide ions are generated through nano electrospray ionization, guided into a mass spectrometer, mass selected, and then guided into a cryogenically held octupole ion trap. Ions are subsequently cooled to their vibrational ground state through collisions with 5 K helium allowing for high resolution IR and UV spectra to be recorded. The IR spectra are highly sensitive to an ion’s conformation, and the well resolved UV spectra provides a means generate conformer specific IR spectra. With the use quantum mechanical calculations, it is possible to calculate the vibrational spectra of candidate structures for comparison with experimental spectra. Strong correlations between theory and experiment allow for unambiguous structural assignments to be made.</p> <p> Structural studies are performed on β-turn motifs and well as salt-bridge geometries. Beta-turns are a commonly occurring secondary structure in peptides and proteins. It is possible to artificially encourage the formation of this secondary structural element through the incorporation of the D-proline (^DP) stereoisomer followed by a gly or ala residue. Interestingly, the L-proline (^LP) stereoisomer is seen to discourage the formation of beta turn structure. Here were probe the inherent conformational preferences of the diastereomeric peptide sequences YA^LPAA and YA^DPAA. The findings agree with solution phase studies, the ^DP sequence is observed to adopt a beta turn however, the ^LP sequence is found to undergo a sterically driven <i>trans</i> à <i>cis</i> isomerization about the proline amide bond. We find the energetics associated with this unfavorable interaction and show the ability to reverse it by proper substitution of Ala₂ for a Gly.</p> <p>The studies directed towards gas phase salt bridges have been limited to single amino acids or dipeptides. Generally, these species are ionized using a metal ion or adducted with water or excess electrons in order to stabilize a zwitterionic motif. Here we take the first look at a salt bridge motif incorporated into polypeptide in order to understand how the solvation from the secondary structure can aid in stabilizing these motifs in non-polar environments. We find a unique salt bridge motif in the YGRAR sequence in which the tyrosine OH acts as a neutral bridge to form a network between the C-terminal arginine and the ion pair formed between the central arginine and C-terminal carboxylate group. This binding motif has not been discussed in literature and appears as an important structural element in non-polar environments as all salt bridge character is lost upon substituting Tyr for Phe. We are the process of mining the PDB for these types of interactions. </p> <p>To better understand how cryo-cooling impacts the resulting population distribution at 10 K we measured the distribution among the two major conformation of the YGPAA ion. This was carried out using population transfer spectroscopy. In this method conformational isomerization is induced vis single conformer infrared excitation. The change in population can be related to the final population distribution at 10 K. With this number, we were able to develop a cooling model to simulate the change in the distribution as a function of cooling. The cooling rates, were experimental established, and the isomerization rates and starting population were theoretically derived through RRKM and thermodynamic calculations. With these parameters and cooling model, we found that the room temperature population distribution is largely preserved. When isomerization events involve breaking a hydrogen bond, they become too slow to complete with the cooling time scale of the experiment, effectively freezing in the room temperature structures. These are important physical parameters to characterize when performing structural studies at 10 K.</p> <p>Finally, we demonstrate a 2-Color IRMPD technique that is able to generate linear spectra at varied temperatures. This is in sharp contrast to traditional IRMPD which results in non-linear and skewed spectra. The importance of generating linear spectra when making structural assignments is highlight by comparing the performance between both techniques. Furthermore, with this technique we show the ability to record the spectra of ion prepared with high internal energies. This provides spectroscopic snapshots of the unfolding events leading to dissociation. Overall, the versatility of this technique to record ground state spectra comparable to IR-UV DR, to record linear spectra at room temperature, and to probe dynamics proves this technique to be useful in the field of ion spectroscopy.</p>

<p>  </p> <p>Sulfonation is a widespread biological reaction catalyzed by a supergene family of enzymes called sulfotransferases (SULTs). SULTs utilize 3’-phosphoadenosine-5’-phospho-sulfate (PAPS) as the universal sulfonate donor to conjugate with a diverse range of endo- and xenobiotic substrates, including neurotransmitters, hormones, and drugs resulting in altering their biological activity. This reaction serves as a major detoxification pathway as conjugation with a sulfonate group renders substrates more hydrophilic and facilitates excretion. Therefore, this process is responsible for reducing the bioavailability of some drugs. In some cases, sulfo-conjugation causes the bio-activation of pro-mutagens and pro-carcinogens, leading to SULTs being risk factors in some cancers. Despite the biological relevance, understanding of this family of enzymes is still scarce. One SULT member that is the focus of the studies described herein is human sulfotransferase 2B1b (SULT2B1b), which had been identified as a potential drug target in prostate cancer. However, the inconsistency in reported kinetic data obtained using radiolabeled assays and the lack of robust assays have become significant limitations for SULT2B1b-targeted drug discovery studies. A label-free assay was developed to bridge this knowledge gap that directly quantifies SULT2B1b sulfonated products. This novel assay utilized high-throughput technology based on Desorption Electrospray Ionization Mass Spectrometry (DESI-MS). Results obtained from the DESI-MS-based assay were compared with those from a fluorometric, coupled-enzyme assay already developed in the Mesecar lab. Both methods provided consistent kinetic data for the reaction of SULT2B1b. Therefore, this novel assay is promising for the application of drug discovery efforts aiming at identifying SULT2B1b inhibitors. The other SULT member studied and described herein is human sulfotransferase 1A1 (SULT1A1), one of humans' most vital detoxifying and drug-metabolizing SULT isoforms that can also be a potential drug target in some cancers. The detailed kinetic mechanism of SULT1A1 was elucidated using steady-state kinetic, product inhibition, dead-end inhibition, and X-crystallographic studies. to gain insights into the role of this enzyme in detoxification, drug metabolism, and the development of inhibitors.</p>

<p>  </p> <p>The development of a novel technique employing the use of a linear quadrupole ion trap mass spectrometer coupled to a Nd:YAG laser and a home-built fast reagent-mixing apparatus is detailed and used to detect the short-lived tetrahedral reaction intermediate of the reaction of acetyl chloride with ethanol in microdroplets. Additionally, tandem and high-resolution mass spectrometry is used to characterize potential precursors for solution-processed metal selenide semiconductors in order to determine a synthetic route to sulfur-free thin films. Lastly, Raman MCR (multivariate curve resolution) spectroscopy is used to study the binding-induced conformational change of 1-bromopropane upon binding to α-cyclodextrin as a model system to examine guest conformational changes upon binding to a host molecule.</p>

<p>  </p> <p>As our ability to make more sensitive measurements increases, we begin to reach for the ultimate measurement sensitivity: a single entity. Mass spectrometry and fluorescence-based methods exist for single entity studies, and through these the intriguing effects of confinement begin to be observed. These mass spectrometry and fluorescence-based techniques are however, often destructive, which precludes measurements over time. The advent of nanoelectrochemical methods, however, allows for the creation of tools that can make measurements inside of confined volumes—a droplet, a cell, <em>etc</em>.—over extended periods of time. While most nanoelectrochemical methods are based on amperometry or voltammetry, potentiometry allows for minimal perturbation of the system’s homeostasis by passing negligible current.</p> <p>To overcome many of the issues involved in these other methods, we design potentiometric tools that can be easily miniaturized to fit within single entities, particularly within single cells. These tools include enzymatic biosensors designed to be minimally influenced by the cell’s O2 or NAD+ levels, as well as a novel reference electrode that can be made to be very low leakage or completely leakless to avoid contamination of the cell by the reference electrode’s internal filling solution. Both of these tools are designed such that they are easily modified to suit a number of different applications, including for use inside non-aqueous solutions.</p>

<p dir="ltr">Spatial heterogeneity is ubiquitous across life and the universe; the same is true for phase-separating pharmaceutical formulations, cells, and tissues. To interrogate these spatially-varying complicated samples, simple analysis techniques such as fluorescence recovery after photobleaching (FRAP) can provide information on molecular transport. Conventional FRAP approaches localize analysis to small spots, which may not be representative of trends across the full field of view.</p><p dir="ltr">Taking advantage of strategies used for structures illumination, an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling. Patterned illumination is used to establish a concentration gradient across a sample by irreversibly photobleaching fluorophores, such as with the simple comb pattern photobleach presented in Chapters 1 and 4. Patterned photobleaching allows spatial Fourier-domain analysis of multiple spatial harmonics simultaneously. In the spatial FT-domain the real-space photobleach signal is integrated into puncta, greatly increasing the signal to noise ratio compared to conventional point-bleach FRAP. The order of the spatial harmonic is directly related to the length-scale of translational diffusion measured, with a series of harmonics accessing diffusion over many length scales in a single experiment. Measurements of diffusion at multiple length scales informs on the diffusion mechanism by sensitively reporting on deviations away from normal diffusion.</p><p dir="ltr">Complementing the physical hardware for inducing patterned illumination, this dissertation introduces novel algorithms for reconstructing spatially-resolved diffusion maps in heterogeneous materials by combining Fourier domain analysis with patterned photobleaching. FT-FRAP is introduced in Chapter 1 for interrogating phase-separating samples using beam-scanning instrumentation for comb-bleach illumination. This analysis allowed disentangling separate contributions to diffusion from normal bulk diffusion and an interfacial exchange mechanism only available due to multi-harmonic analysis. The introduction of a dot-array bleach pattern using widefield microscopy is presented in Chapter 2 for high-throughput detection of mobility in simple binary systems as well as for segmentation in phase-separating pharmaceutical formulations. The analysis becomes more complicated as more components are added to the system such as a surfactant. Introduced in chapter 3, FT-FRAP with dot-array photobleaching was shown to be useful for characterizing diffusion of phase-separating micro-domain smaller than a single pixel of the camera. Supported by simulations, a biexponential fitting model was developed for quantification of diffusion by multiple species simultaneously. Chapter 4 introduces imaging inside of 3D particles comprised of an active pharmaceutical ingredient (API) in microencapsulated agglomerates which exhibited strong interfacial exchange. Multi-photon excited fluorescence enabled imaging a small focal volume within the particles.</p>

<p>The research described in this dissertation focuses on several areas: developing analytical methods to distinguish structural isomers, identifying the chemical compositions of aviation fuels and evaluating the effectiveness of organic dopants to swell aircraft o-ring seals. Chapter 2 discusses fundamental aspects of mass spectrometry, and ionization methods and the instrumentation used to complete this research. </p> <p>Chapter 3 discusses and compares two activation methods used to distinguish ionized structural isomers. Ionized naphthene-containing aromatic structural isomers were subjected to collision-activated dissociation (CAD) in an ion trap (ITCAD) and to medium-energy collision-activated dissociation (MCAD) in an octupole collision cell, both in the energy-resolved mass spectrometry mode (ERMS). MCAD was shown to be superior over ITCAD at the structural differentiation of the ionized isomers. </p> <p>Determination of the chemical compositions of petroleum-based jet and diesel fuels, potential alternative fuels and fuel blending components by using a GCxGC/(EI)TOF MS is discussed in chapter 4. The ability to determine the chemical compositions of fuels and to correlate the identified compounds and their concentrations to the physical and chemical properties and aircraft performance of the fuels is vital for the development of future resilient, alternative fuels. The chemical compositions of petroleum-based fuels were found to be different from potential alternative fuels.</p> <p>Chapter 5 discusses the effectiveness of aromatic and nonaromatic compounds in swelling air craft o-ring seals, which prevents leaks in the fuel circulation systems. The aim of this study was to identify aromatic and nonaromatic compounds that most effectively swell o-ring seals. Steric effects were shown to decrease the efficiency of the compounds to swell seals. Ethylbenzene and indane were found to swell o-ring seals more effectively than any other compounds studied, including a currently approved alternative fuel. </p>

Mass spectrometry is a versatile analytical tool for chemical and biomolecule identification, quantitation, and structural analysis. Tandem mass spectrometry further expands the applications of mass spectrometry, making it more than a mere detector. With tandem mass spectrometry, the mass spectrometer is capable of probing reaction mechanisms, monitoring reaction processes, and performing fast analysis on complex samples. In tandem mass spectrometry, after activation the precursor ions fragment into small fragment ions through one or more pathways, which are affected by the ion’s inherit property, the ion type, and the activation method. To obtain complementary information, one can alter the fragmentation pathway by changing the ion via ion charge manipulation and covalent modification to the ion. Gas-phase ion/ion reactions provide an easy approach to changing ion type and facile modification to the analyte ions. It has been extensively used for spectrum simplification and analyte structural studies. In this dissertation, ion/ion reaction facilitated mass spectrometry methods are studied, and explorations into the method development involving front-end mass spectrometer are discussed.<br>The first work demonstrates a special rearrangement reaction for gas-phase Schiff-base-modified peptides. Gas-phase Schiff-base modification of peptides has been applied to facilitate the primary structural characterization via tandem mass spectrometry. A major or minor fragment pathway related to the novel rearrangement reaction was observed upon in-trap collisional activation of the gas-phase Schiff-base-modified peptides. The rearrangement reaction involves the imine of the Schiff base and a nucleophile present in the polypeptide. The occurrence of the rearrangement reaction is affected by several factors, such as ion polarity, identity of the nucleophile in the peptide (e.g., side chains of lysine, histidine, and arginine), and the position of the nucleophile relative to the imine. The rearrangement reaction does not affect the amount of structural information that can be obtained by collisional activation of the Schiff-base-modified peptide, but when the rearrangement reaction is dominant, it can siphon away signal from the structurally diagnostic processes.<br>Efforts have also been put into the method development of peptide and protein aggregation detection via electrospray ionization mass spectrometry (ESI-MS). People have studied peptide and protein aggregation processes to understand the mechanism of amyloid-related diseases and to control the quality of the peptide and protein pharmaceuticals. ESI-MS is suitable for solution aggregation studies because of its compatibility with solution samples and the straightforward result of the analyte’s oligomeric state on the mass spectrum. However, peak overlap issue and nonspecific aggregation in the ESI process can obscure the result. Here, the application of proton transfer ion/ion reaction to the analyte has been found useful to reduce or eliminate the peak overlap issue. A statistical model based on Poisson statistics has been proposed to deal with the ESI-induced nonspecific aggregation in the droplet and to differentiate the solution-phase aggregation from the droplet-induced aggregation. Factors that affect the accuracy of the statistical model have been discussed with MATLAB simulations.<br>In the era of biological system studies, sample complexity is a challenge every analytical chemist has to face. The analysis of complex sample can be facilitated by the combination of separation techniques outside the mass spectrometer (such as differential mobility spectrometry (DMS)) and ion structure probing techniques inside the mass spectrometer (such as tandem mass spectrometry and gas-phase ion/ion reactions). Here the coupling method between DMS and ion/ion reaction is developed and tested with model peptide systems to demonstrate its possible application in complex sample characterization such as isomer identification.<br>

<p>   </p> <p>Ambient Mass Spectrometry Imaging (MSI) is a powerful analytical tool in biomedical research that enables simultaneous label-free spatial mapping of hundreds of molecules in biological samples under native conditions. Nanospray desorption electrospray ionization (nano-DESI) is an emergent ambient MSI technique developed in 2010 that uses localized liquid extraction of molecules directly from surfaces. Like other liquid-extraction based techniques, nano-DESI relies on gentle removal of molecules from surfaces and soft ionization. High sensitivity and spatial resolution, versatility of the solvent composition, which may be used to tailor the extraction and ionization of selected molecules, quantification capabilities at the single-pixel level as well as compensation for matrix effects by adding a known standard to the solvent, and online derivatization are key features of nano-DESI MSI that position it as a unique analytical tool for studying biological systems. </p> <p>Despite the advantages that nano-DESI provides, there are still challenges associated with the structural characterization, extraction, and detection of certain molecular classes. Therefore, my dissertation research has focused on addressing these analytical challenges by developing innovative approaches that substantially enhance the performance of the nano-DESI technique in the study of complex biological systems. </p> <p>In this thesis, a systematic study of the solvent composition is carried out to aid in the detection of neutral lipids such as triglycerides thereby expanding the molecular coverage of nano-DESI experiments. Taking advantage of the versatility of the solvent composition, I developed an approach for the online derivatization of unsaturated lipids into lipid hydroperoxides using the reaction of singlet oxygen with C=C bonds. This method further expands the specificity of nano-DESI MSI by enabling the detection and imaging of positional lipid isomers. To aid in the analysis of complex mixtures and provide additional structural information in the form of collision cross sections, coupling of nano-DESI with a drift-tube ion mobility spectrometry is also reported along with examples of the powerful capabilities of this platform. Lastly, nano-DESI MSI is used to address the complexity in the analysis of individual skeletal muscle fibers. This collaborative project involves the development of a robust image registration approach of immunofluorescence imaging and high-spatial resolution nano-DESI MSI to obtain accurate chemical maps specific to each fiber type. The developments described in this thesis are key to understanding the dynamic metabolic processes on a molecular level with an unprecedented specificity and sensitivity.</p> <p>  </p>

Mass spectrometry of negative ions is a convenient method for generating, isolating, and analyzing reactive intermediates that would otherwise be too short lived to detect. This ion approach is especially useful for studying the chemical properties of radicals. In this work, a negative charge-carrying group was attached to lignin model compounds and combined with collision-induced dissociation (CID) to generate and characterize radical species involved in the primary pyrolysis of lignin. The charge-tag served to increase the sensitivity of the model compounds using electrospray ionization mass spectrometry (ESI-MS) and promoted charge-remote fragmentations (CRF) upon being collisionally activated. The resulting product ions were comparable to the primary pyrolysis products of lignin; thus, CID-CRF proved to be an effective way of identifying the mechanisms by which lignin decomposes in the gas phase. <br><div><br></div><div>Additionally, this dissertation includes a review of nitrene anions. Nitrene anions are another class of reactive intermediates protected by an electron that provide a means for studying the corresponding neutral molecules via electron photodetachment spectroscopy and photoelectron spectroscopy. The added electron makes it possible for protected nitrene anions to be manipulated by external electric and magnetic fields of a mass spectrometer. Nitrene anions also display their own unique reactivities as reagents, which have been investigated using ion/molecule reactions. Mass spectrometry of negative ions has thereby provided information on the electronic states, reactivities, and thermochemical properties of nitrene intermediates.</div>